The continued prevalence of hospital-acquired infections and concomitant clinical and epidemiological interest in typing isolates have resulted in many large clinical laboratories using molecular epidemiological methods to investigate the relatedness of clinically isolated microorganisms. In the Microbiology Service, we are currently evaluating different approaches to molecular strain typing, including plasmid analysis, restriction endonuclease analysis of genomic DNA, pulsed-field gel electrophoresis (PFOE) of genomic DNA, and typing methods based on the polymerase chain reaction. The goal of this project is to develop a coordinated battery of typing methods that can be used to provide information on a wide variety of clinically significant microorganisms. This will permit us to respond promptly in potential outbreak or nosocomial spread situations. Our technical development of these methods includes simplifying procedures so that they can be done rapidly, and thereby be of greater epidemiological use. The use of molecular typing methods greatly expands the species of organisms for which we will be able to offer typing. Additional uses of typing availability will be to look at the relatedness of species common to particular patient populations. Thus far, the use of plasmid analysis and PFGE has proven reliable and sufficiently discriminatory for staphylococci. PFGE will also be suitable for a variety of gram-negative rods, such as Serratia. PFOE, however, is slow and labor-intensive, often needing to be repeated. We have found that, for some organisms, getting an adequate number of bands for sufficient discrimination is difficult. Our recent work with random amplified polymorphic DNA (RAPD) assays may prove to be of greater use for these organisms, such as has been the case with Roseomonas and Burkholderia species. We have already used our molecular typing procedures to investigate several questions of either patient-to- patient spread, or common-source acquisition of pathogens, and by working in conjunction with the Hospital Epidemiology Service have been able to quickly resolve those issues. Our current developmental concerns are to define optimal procedures for each group of significant pathogens so that we can respond reliably when needed. At present we can reliably type strains of staphylococci, enterococci, Pseudomonas, Roseomonas, Serratia, and Burkholderia by either PFGE or RAPD. We are planning to enhance our ability to utilize RAPD in place of PFGE for preliminary results, since RAPD can be done much more rapidly and often more reliably and definitively than PFGE. We are also working on setting up a data repository for PFOE and RAPD patterns in order to be able to store, retrieve, and manipulate the strain typing data that we generate. This will allow us to retrieve hospital pattern data, e.g., for detecting trends in the distribution of antibiotic resistant strains, as well as be able to follow a particular patient or set of patients in a longitudinal fashion, looking at their colonization/ infection patterns. We are particularly interested at looking at specific patient populations that have recurrent infections to determine the patterns of colonization, infection, and relapse re-infection that can now be determined by these methods.